Knut Dahl-Jørgensen

Syntaxin-4, a beta cell plasma membrane-anchored protein, attaches to beta cell granules with two spatially-located nearby proteins, facilitating the fusion of beta cell-docked granules to the plasma membrane, followed by exocytotic release of insulin and remaining granule contents. During type 1 diabetes (T1D) and its preceding phase, it is unclear whether syntaxin-4 levels decline in residual beta cells, thereby contributing to impaired insulin release. The level of syntaxin-4 in insulin-positive regions of islets was determined following immunofluorescence staining of human pancreatic sections, with co-immunofluorescence staining for insulin and for glucagon by immunoperoxidase. Random islets from non-diabetic autoantibody-negative (Group 1; n = 7), non-diabetic autoantibody-positive (Group 2; n = 6), new-onset T1D (Group 3; n = 4) and long-term T1D (Group 4; n = 10) donors were quantified for syntaxin-4 levels. In groups 1 and 2, inter-islet staining intensities for syntaxin-4, although variable across individual donors, were similar overall between the groups, with lower intensities in group 3. In group 4, 3 donors with 0.58–1.5 years of T1D showed mean intensities comparable to groups 1 and 2, whereas donors with 4–7 years of T1D showed declining intensities. In general, the average syntaxin-4 intensities in the two diabetic groups were lower than those in the non-diabetic groups. The expression of syntaxin-4 in insulin-positive islet cells showed marked differences in intensity among most diabetic donors and autoantibody-positive and -negative non-diabetic donors. In some long-term diabetic donors, syntaxin-4 was also present in insulin- and glucagon-negative cells. During protracted T1D, reduced syntaxin-4 in islets may impair insulin release.

During type 1 diabetes (T1D), oxidative stress in beta cells may cause early beta cell dysfunction and initiate autoimmunity. Mouse islets express lower levels of reactive oxygen species (ROS) clearing enzymes, such as glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase than several other tissues. It remains unclear if human beta cells show a similar deficiency during T1D or exhibit a higher degree of intrinsic resistance to oxidative stress. We compared islet cell distributions and determined graded intensities of glutathione peroxidase1 (GPX1), a key enzymatic mediator involved in detoxifying hydrogen peroxide, by applying combined immunohistochemistry for GPX1, insulin and glucagon, in pancreatic sections from new-onset T1D (group 1), non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic (group 4) donors. Islets from all study groups demonstrated either uniform but graded staining intensities for GPX1 in almost all islet cells or strong staining in selective islet cells with weaker intensities in the remaining cells. GPX1 was present in selective glucagon cells and insulin cells, including in cells negative for both hormones, with stronger intensities in a higher percentage of glucagon than insulin cells. It was absent in a higher percentage of beta cells than glucagon cells independent of disease or autoantibody positivity. We conclude that a proportion of human beta cells and glucagon cells express GPX1 but show heterogeneity in its distribution and intensities, independent of disease or autoantibody status. Our studies highlight important differences in the expression of GPX1 in islet cell-types between mice and humans.

Aims/hypothesis: The organisation and cellular architecture of islets of Langerhans are critical to the physiological regulation of hormone secretion but it is debated whether human islets adhere to the characteristic mantle–core (M-C) structure seen in rodents. It is also unclear whether inherent architectural changes contribute to islet dysfunction in type 1 diabetes, aside from the loss of beta cells. Therefore, we have exploited advances in immunostaining, spatial biology and machine learning to undertake a detailed, systematic analysis of adult human islet architecture in health and type 1 diabetes, by a quantitative analysis of a dataset of >250,000 endocrine cells in >3500 islets from ten individuals. Methods: Formalin-fixed paraffin-embedded pancreatic sections (4 μm) from organ donors without diabetes and living donors with recent-onset type 1 diabetes were stained for all five islet hormones and imaged prior to analysis, which employed a novel automated pipeline using QuPath software, capable of running on a standard laptop. Whole-slide image analysis involved segmentation classifiers, cell detection and phenotyping algorithms to identify islets, specific cell types and their locations as (x,y)-coordinates in regions of interest. Each endocrine cell was categorised into binary variables for cell type (i.e. beta or non-beta) and position (mantle or core). A χ² test for independence of these properties was performed and the OR was considered to estimate the effect size of the potential association between position and cell type. A quantification of the M-C structure at islet level was performed by computing the probability, r, that the observed number of non-beta cells in the mantle is due to a random arrangement. The distribution of the r values for the islets in the study was contrasted against the r values of a digital population of equivalent randomly arranged islets, termed digital siblings. Both distributions of r values were compared using the earth mover’s distance (EMD), a mathematical tool employed to describe differences in distribution patterns. The EMD was also used to contrast the distribution of islet size and beta cell fraction between type 1 diabetes and control islets. Results: The χ² test supports the existence of a significant (pr values of the observed islets and the digital siblings was emd-1d=0.10951 (0r value, thus supporting the M-C hypothesis. The bidimensional distribution (beta cell fraction vs size) of islets showed a distance emd-2d=0.285 (0<emd-2d<2) between the control and type 1 diabetes islets. The suffixes ‘-1d’ and ‘-2d’ are used to distinguish the comparison between the distribution of one and two variables. Conclusions/interpretation: Using a novel analysis pipeline, statistical evidence supports the existence of an M-C structure in human adult islets, irrespective of type 1 diabetes status. The methods presented in the current study offer potential applications in spatial biology, islet immunopathology, transplantation and organoid research, and developmental research. Graphical

OBJECTIVE: In the Diabetes Virus Detection and Intervention trial, antiviral treatment with pleconaril and ribavirin decreased the decline, compared with placebo, in endogenous C-peptide 1 year after diagnosis of type 1 diabetes (T1D) in children and adolescents. This article reports the results 2 and 3 years after diagnosis. RESEARCH DESIGN AND METHODS: This was a multicenter, randomized, placebo-controlled (1:1) trial of 96 children and adolescents aged 6–15.9 years newly diagnosed with T1D. Antiviral treatment (pleconaril and ribavirin) or placebo was given for 6 months from diagnosis, and participants were followed for 3 years. The primary outcome was residual C-peptide secretion, reported as the area under the curve (AUC), assessed by 2-h mixed-meal tolerance test. Secondary outcomes included insulin doses and HbA1c. RESULTS: At the 3-year follow-up, 75 participants attended. At 2 years, the mean ± SD AUC for C-peptide in the placebo group was 0.27 ± 0.33 compared with 0.34 ± 0.37 in the pleconaril and ribavirin group. After 3 years, the AUC had decreased to 0.17 ± 0.23 and 0.25 ± 0.34, respectively. There was no statistically significant difference between the groups. The groups were also comparable with regard to secondary end points. CONCLUSIONS: The decreased reduction in C-peptide levels after antiviral treatment is no longer present after 2 or 3 years. Further investigations are needed to explore options to use antiviral treatment in the prevention and treatment of T1D.

OBJECTIVE: Young women with type 1 diabetes are at risk to develop eating disorders (ED). We evaluated a novel ED prevention program in a multinational randomized controlled trial. RESEARCH DESIGN AND METHODS: Women (14–35 years old) with type 1 diabetes were randomized to virtual Diabetes Body Project groups or educational control. Outcomes were assessed at pretest and posttest (1–2 weeks after intervention). RESULTS: Compared with educational controls (n = 146), participants in the Diabetes Body Project (n = 147) showed significant improvements (all P < 0.05), with small Cohen’s d effect sizes for ED symptoms (d = −0.30, 95% CI −0.06, −0.69) (primary outcome), diabetes distress (d = −0.42), quality of life (d = 0.39) and dietary restraint (d = −0.31), and medium effect sizes for diabetes-specific disordered eating behaviors (d = −0.70), body dissatisfaction (d = −0.59), and pursuit of thin appearance ideal (d = −0.56). CONCLUSIONS: The Diabetes Body Project produced significantly larger acute effects with small to medium effect sizes compared with educational controls and has potential for broad implementation.

During type 1 diabetes (T1D), oxidative stress in beta cells may cause early dysfunction and initiate autoimmunity. Mouse islets express lower levels of reactive oxygen species (ROS) clearing enzymes, glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase in comparison with several other tissues. It remains unclear if human beta cells show a similar deficiency, particularly during T1D or show a higher degree oxidative resistance. We compared islet cell distributions and determined graded intensities of glutathione peroxidase1 (GPX1), a key enzyme which detoxifies hydrogen peroxide, by applying combined immunohistochemistry for GPX1, insulin and glucagon, in pancreatic sections from new-onset T1D (group 1), non-diabetic autoantibody-negative (group 2), non-diabetic autoantibody-positive (group 3) and long-term diabetic (group 4) donors. Islets from all study groups demonstrated either uniform but graded staining intensities for GPX1 in almost all islet cells or strong staining in selective islet cells with weaker intensities in the remaining cells. GPX1 was present in selective glucagon and in insulin cells also and in cells negative for both hormones, with stronger intensities in a higher percentage of glucagon than insulin cells. The enzyme was absent in a higher percentage of beta cells than glucagon cells independent of disease or autoantibody positivity. We conclude that a proportion of human beta cells and glucagon cells express GPX1 but show heterogeneity in its distribution and intensities, independent of disease or autoantibody status. Pharmacologic attempts to boost GPX1 level in human beta cells during early T1D or in isolated islets for transplantation may offer clinical benefits.

Aims/Hypothesis: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system. Material and methods: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes). Results: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type 1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes. Conclusions: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed.

Previous studies showed a low-grade enterovirus infection in the pancreatic islets of patients with newly diagnosed type 1 diabetes (T1D). In the Diabetes Virus Detection (DiViD) Intervention, a phase 2, placebo-controlled, randomized, parallel group, double-blind trial, 96 children and adolescents (aged 6–15 years) with new-onset T1D received antiviral treatment with pleconaril and ribavirin (n = 47) or placebo (n = 49) for 6 months, with the aim of preserving β cell function. The primary endpoint was the mean stimulated C-peptide area under the curve (AUC) 12 months after the initiation of treatment (less than 3 weeks after diagnosis) using a mixed linear model. The model used longitudinal log-transformed serum C-peptide AUCs at baseline, at 3 months, 6 months and 1 year. The primary endpoint was met with the serum C-peptide AUC being higher in the pleconaril and ribavirin treatment group compared to the placebo group at 12 months (average marginal effect = 0.057 in the linear mixed model; 95% confidence interval = 0.004–0.11,P = 0.037). The treatment was well tolerated. The results show that antiviral treatment may preserve residual insulin production in children and adolescent with new-onset T1D. This provides a rationale for further evaluating antiviral strategies in the prevention and treatment of T1D. European Union Drug Regulating Authorities Clinical Trials identifier:2015-003350-41.

Aims/hypothesis: Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures. Methods: Six recent-onset type 1 diabetes patients (age 24–35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43–83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1–4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5′ untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies. Results: Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein–Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5′ untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1). Conclusions/interpretation: Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents. Graphical